Hearts Of Anesthetized Rats Research Articles

Oxidative stress inactivates ecto-5'-nucleotidase by inhibiting protein kinase C in rat hearts in vivo

Examined in the present study, allopurinol are xanthine oxidase inhibitors for use in rat hearts in vivo dialysis technology and ventricular myocardial intersitial adenosine production can increase. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts and the tissue in the vicinity of the dialysis was perfused with Tyrode's solution containing adenosine 5'-monophosphate (AMP) through the dialysis probe at a rate of 1.0ml/min to assess the activity of ecto-5'-nucleotidase. Allopurinol (10μM) significantly increased the level of adenosine in rat heart dialysate (n=6, p<0.05), which was inhibited by chelerythrine, 10μM, an antagonist of protein kinase C (PKC). Another free radical scavenger, coenzyme Q10 (CoQ10, 100μM) or ascorbic acid (Vitamin C; 100μM) also increased adenosine production. In addition, allopurinol enhanced the diacylglycerol (DAG; 50μM)-induced also increases in adenosine production by 71.5±12.0% (n=6, P<0.05), to a level significantly (P<0.05) greater than the increase caused by DAG alone (33.0±10.6%). In the presence of allopurinol (10μM), a marked elevation of AMP-primed dialysate adenosine in ischemia/reperfused rat hearts was observed. Free radical generation may suppress adenosine production via activation of PKC. The results suggest that oxidative stress may cause inactivation of nucleotidase, adenosine production in rat heart.

The cardioprotective effect of different doses of vasopressin (AVP) against ischemia–reperfusion injuries in the anesthetized rat heart

The aim of the present study was to investigate the protective effect of various doses of exogenous vasopressin (AVP) against ischemia–reperfusion injury in anesthetized rat heart. Anesthetized rats were randomly divided into seven groups ( n = 4–13) and all of them subjected to prolonged 30 min regional ischemia and 120 min reperfusion. Group I served as saline control with ischemia, in treatment groups II, III, IV and V, respectively different doses of AVP (0.015, 0.03, 0.06 and 1.2 μg/rat) were infused within 10 min prior to ischemia, in group VI, an AVP-selective V1 receptor antagonist (SR49059, 1 mg/kg, i.v.) was administrated prior to effective dose of AVP injection and in group VII, SR49059 (1 mg/kg, i.v.) was only administrated prior to ischemia. Various doses of AVP significantly prevented the decrease in heart rate (HR) at the end of reperfusion compared to their baseline and decreased infarct size, biochemical parameters [LDH (lactate dehydrogenase), CK-MB (creatine kinase-MB) and MDA (malondialdehyde) plasma levels], severity and incidence of ventricular arrhythmia, episodes and duration of ventricular tachycardia (VT) as compared to control group. Blockade of V1 receptors by SR49059 attenuated the cardioprotective effect of AVP on ventricular arrhythmias and biochemical parameters, but partially returned infarct size to control. AVP 0.03 μg/rat was known as effective dose. Our results showed that AVP owns a cardioprotective effect probably via V1 receptors on cardiac myocyte against ischemia/reperfusion injury in rat heart in vivo.

Oxytocin protects rat heart against ischemia–reperfusion injury via pathway involving mitochondrial ATP-dependent potassium channel

Cardiac preconditioning represents the most potent and consistently reproducible method of rescuing heart tissue from undergoing irreversible ischemic damage. One of the major goals of the current cardiovascular research is to identify a reliable cardioprotective intervention that can salvage ischemic myocardium. The aim of the present study is to evaluate the oxytocin (OT)-induced cardioprotection and the signaling pathway involved with mitochondrial ATP-dependent potassium (mitoKATP) channel in the anesthetized rat heart. Animals were divided into six groups ( n = 6): (1) IR; hearts were subjected to 25 min ischemia and 120 min reperfusion, (2) OT; oxytocin was administered (0.03 μg/kg i.p.) 25 min prior to ischemia, (3) ATO+OT; atosiban (ATO) was used as an OT-selective receptor antagonist (1.5 μg/kg i.p.) 10 min prior to OT administration, (4) ATO; atosiban was used 35 min prior to ischemia, (5) 5HD + OT; 5-hydroxydecanoic acid (5HD) was used as a specific inhibitor of mitoKATP channel (10 mg/kg i.v.) 10 min prior to OT administration, (6) 5HD; 5HD was used 35 min prior to ischemia. Then infarct size, ventricular arrhythmia and creatine kinase-MB isoenzyme (CK-MB) plasma level were measured. Hemodynamic parameters were recorded throughout the experiment. OT administration significantly decreased infarct size, CK-MB plasma level, severity and incidence of ventricular arrhythmia as compared to IR group. Administration of atosiban and 5HD abolished the cardiopreconditioning effect of OT. This study demonstrates that cardioprotective effects of OT are mediated through opening the mitoKATP channels.

Noradrenaline Protects In Vivo Rat Heart Against Infarction and Ventricular Arrhythmias Via Nitric Oxide and Reactive Oxygen Species

Our previous study showed that pretreatment with noradrenaline via opening of the mitochondrial ATP-sensitive potassium channel protects myocardium against ischemia/reperfusion injuries. We have hypothesized that production of nitric oxide (NO) and generation of reactive oxygen species (ROS) are involved in noradrenaline-induced cardioprotection in rat heart. All anesthetized rats underwent 25 min of regional ischemia followed by 120 min of reperfusion. Animals were randomized to receive one of the following treatment: saline, noradrenaline (2 μg/kg, i.v.), noradrenaline plus prazosin (an α(1)-adrenoceptor blocker, 0.5mg/kg, i.v.), noradrenaline plus L-NAME (a nonspecific NOS inhibitor, 10mg/kg, i.v.), noradrenaline plus tempol (a membrane-permeable radical scavenger, 30 mg/kg, i.v.), Prazosin alone, only L-NAME and tempol alone. Infarct size (% of risk area) was reduced from 49.6 ± 2.4 in saline-control group to 18.2 ± 1.5 in noradrenaline preconditioned group. Administration of prazosin, L-NAME, or tempol prior to noradrenaline injection abolished the observed cardioprotection of noradrenaline (45.5 ± 3, 41.7 ± 4.5 and 38.7 ± 5.4, respectively) and restored infarct size to saline-control rats' level. Incidences and severity of ventricular arrhythmia during ischemia and early reperfusion significantly decreased in noradrenaline preconditioned group compared with saline-control group. This cardioprotective effect of noradrenaline against ventricular arrhythmia was abrogated by administration of prazosin, L-NAME, or tempol. Cardioprotection effect of the α(1)-adrenoceptor stimulation by noradrenaline was inhibited by L-NAME or tempol in anesthetized rat heart.

Chemical preconditioning effect of 3-nitropropionic acid in anesthetized rat heart

Short ischemic episodes increase tolerance against subsequent severe ischemia in the heart. Nitropropionate (3-NP), an irreversible inhibitor of succinic dehydrogenase of the mitochondrial complex II, was shown to induce protective effect against ischemic brain injury. The aim of this study was to investigate the possible protective effect of 3-NP on regional ischemia in preconditioned rat heart in vivo. Hearts were assigned into three groups: first, in order to induce ischemic preconditioning (IP) 5 min ischemia separated by 10 min reperfusion protocol was used; second, non-preconditioned group was used as control; and third, 3-NP (20 mg/kg, i.p.) was injected 3 h before the surgical procedure in order to induce chemical preconditioning. In all these groups, 30 min regional ischemia was followed by 60 min reperfusion. Infarct size, bax expression, number of ventricular ectopic beats (VEB), duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) were significantly decreased in ischemic preconditioning and 3-NP pretreatment groups, whereas bcl-2 values were not markedly changed in these groups during occlusion period. These results showed that in the anesthetized rat heart 3-NP induced chemical preconditioning by decreasing infarct size, number of VEB, duration of VT and VF. Protective effect is associated with via decreased production of bax protein expression.

Remifentanil Preconditioning Confers Cardioprotection via Cardiac κ- and δ-Opioid Receptors

Remifentanil preconditioning (RPC) reduces the infarct size in anesthetized rat hearts, and this effect seems to be mediated by all three types of opioid receptors (ORs). Because there is evidence of only kappa- and delta- but not mu-ORs in the rat heart, the authors investigated whether RPC confers cardioprotection via cardiac kappa- and delta-OR as well as via extracardiac mu-OR agonist activity. The authors also investigated the involvement of signaling mechanisms, namely protein kinase C and mitochondrial adenosine triphosphate-sensitive potassium (KATP) channels. The hearts of male Sprague-Dawley rats weighing 190-210 g were removed, mounted on a Langendorff apparatus, and perfused retrogradely at 100 cm H2O with Krebs-Ringer's solution. All hearts were subjected to 30 min of ischemia and 2 h of reperfusion. The study consisted of three series of experiments on the effect of ischemic preconditioning or RPC (10, 50, and 100 ng/ml remifentanil) after blockade of OR subtypes (delta-OR antagonist naltrindol, kappa-OR antagonist nor-binaltorphimine, and mu-OR antagonist CTOP). The involvement of protein kinase C or the KATP channel in the cardioprotection of RPC was also investigated using specific blockers in each group. RPC was produced by three cycles of 5-min perfusion of remifentanil in Krebs-Ringer's solution interspersed with a 5-min reperfusion with Krebs solution only. Infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining. Infarct size as a percentage of the area at risk was significantly reduced after RPC from 51.9 +/- 5.0% (control, n = 8) to 36.2 +/- 10.0% (100 ng/ml RPC, n = 8, P < 0.01). This effect was stopped by pretreatment with naltrindol (52.3 +/- 5.2%) and nor-binaltorphimine (43.5 +/- 6.0%) but not CTOP (37.1 +/- 6.0%). Chelerythrine and GF109203X, both protein kinase C inhibitors, abolished the effects of RPC or ischemic preconditioning on infarct size as a percentage of area at risk. 5-Hydroxydecanoate (a selective mitochondrial KATP channel blocker) also abolished the cardioprotection of RPC and IPC, but HMR-1098 (a selective inhibitor of the sarcolemmal KATP channel) did not. Cardiac delta- and kappa- but not mu-ORs mediate the cardioprotection produced by RPC. Both protein kinase C and the mitochondrial KATP channel were involved in this effect.

Norepinephrine evoked by potassium depolarization increases interstitial adenosine concentration via activation of ecto-5'-nucleotidase in rat hearts.

We examined whether the increase of the extracellular potassium ion concentration, [K(+)](o), can increase the production of interstitial adenosine in the ventricular myocardium, with the use of microdialysis techniques in in situ rat hearts. A microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts, and the tissue in the vicinity of the dialysis was perfused with Tyrode's solution containing AMP through the dialysis probe at a rate of 1.0 microl/min to assess the activity of ecto-5'-nucleotidase. When the K(+) concentration of the perfusate ([K(+)](o)) was increased stepwise from 5.4 mM (control) to up to 140.4 mM, the level of dialysate adenosine significantly increased, in a [K(+)](o)-dependent manner. The presence of CsCl or BaCl(2) (20 mM), which markedly depolarized the resting potential, significantly increased the level of adenosine in the dialysate. Equivalent increases in the osmotic concentration of the perfusate, made by adding sucrose (270 mM), did not change the dialysate adenosine concentration. Introduction of high [K(+)](o) (140.4 mM) significantly increased the level of norepinephrine (NE) in the dialysate, and this increase was abolished in the reserpinized rats hearts. In the presence of an antagonist of alpha(1)-adrenoceptor (prazosin, 50 microM) or protein kinase C (PKC) (chelerythrine, 10 microM) and in reserpinized rats, an introduction of high [K(+)](o) failed to increase the AMP-primed dialysate adenosine concentration. We conclude that high [K(+)](o)-induced NE release from sympathetic nerve terminals increases adenosine by stimulating the PKC-ecto-5'-nucleotidase cascade through alpha(1)-adrenoceptors.

An increase of the native interstitial adenosine concentration during histidine application.

We examined whether histidine can increase the production of interstitial adenosine via noradrenaline (NA) release-mediated activation of ecto-5'-nucleotidase in the ventricular myocardium, with use of microdialysis techniques in in situ rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts and the tissue was perfused with Tyrode's solution containing adenosine 5'-monophosphate (AMP) through the dialysis probe at a rate of 1.0 microl/min. Adenosine in the dialysate collected during perfusion with Tyrode's solution containing 100 microM AMP (through the probe) originated from the hydrolysis of AMP catalyzed by endogenous ecto-5'-nucleotidase, so that the level of adenosine reflected the activity of ecto-5'-nucleotidase in this tissue. In the presence of NA (10 microM), histidine, a scavenger of highly active singlet oxygen (1O2), significantly increased concentration of adenosine. Histidine (5-50 mM) increased the level of AMP-primed dialysate adenosine in a concentration-dependent manner. When histidine (25 mM) was infused to rat myocardium, small increase in the levels of adenosine were observed. However, when corresponding experiments were performed with NA (10 microM)-pretreated animals, a marked elevation of the level of adenosine in rat heart dialysate was obtained. To confirm the possible mechanism of interaction between 1O2 and NA, we examined the effect of histidine in ischemic-reperfused rat hearts. In the presence of histidine (25 mM), a marked elevation of NA and adenosine was observed. However, when corresponding experiments were performed with reserpinized rat hearts, the elevation of both NA and adenosine was not observed in ischemia-reperfused rat hearts. These results indicate that histidine increases interstitial adenosine concentration via NA release-mediated activation of ecto-5'-nucleotidase.

Reperfusion arrhythmias in the murine heart: their characteristics and alteration after ischemic preconditioning.

In this study, we examined the features of reperfusion arrhythmias and the effect of preconditioning (PC) in the mouse for future application of genetically engineered mice to study mechanisms of this type of arrhythmia. Under pentobarbital anesthesia, reperfusion arrhythmias were induced by temporary occlusion of the left anterior descending coronary artery and reperfusion in BALB/c AJc1 mice. In the first protocol, the coronary artery was occluded for periods ranging from 2 to 15 min and then reperfused. In the second protocol, hearts were preconditioned with 2- or 3-min ischemia and 5-min reperfusion prior to the 5 min of coronary occlusion. An electrocardiogram was recorded throughout the experiment, and arrhythmias were diagnosed according to the Lambeth Convention criteria. The incidences of reperfusion-induced ventricular tachycardia (VT) in hearts that received 2, 3, 5, 10 and 15-min ischemia (n = 10 approximately 14) were 0, 9, 73, 55 and 30 %, respectively. Ventricular fibrillation (VF) was not observed upon reperfusion regardless of the ischemia duration. PC with 2-min ischemia and with 3-min ischemia (n = 10 for each PC) reduced the incidences of reperfusion VT after 5-min ischemia to 40% and 10%, respectively. However, in mice that developed reperfusion VT, the VT duration was similar to that in non-preconditioned controls, ranging from 1 to 16 s. These results suggest that the relationship between ischemia duration and incidence of VT upon subsequent reperfusion is "bell shaped" and that PC has anti-arrhythmic effects in the mouse, as it does in anesthetized rat hearts. However, there appear to be differences in the incidence of reperfusion-induced VF and the duration of reperfusion VT between these species. Thus, the present murine preparation appears to be a useful model for studying the mechanism of reperfusion VT and PC, though it does not share all of the features of reperfusion arrhythmias with the anesthetized rat preparation.

α-Tocopherol acetate significantly suppressed the increase in heart interstitial 8-hydroxydeoxyguanosine following myocardial ischemia and reperfusion in anesthetized rats

The effect of α-tocopherol acetate, an aqueous form of α-tocopherol, on the increase in heart interstitial 8-hydroxydeoxyguanosine (8-OH-dG) levels following myocardial ischemia/reperfusion was investigated. A microdialysis probe was implanted in the left ventricular interstitial space of anesthetized rat hearts. Myocardial ischemia was induced by ligating the left anterior descending coronary artery. Levels of 8-OH-dG in microdialysates were analyzed via an on-line high-performance liquid chromatography system equipped with an electrochemical detector. The 8-OH-dG levels significantly increased (maximum 3.6-fold of increase relative to basal value) during the 60-min reperfusion stage following a 20 min ischemia. Administration of α-tocopherol acetate (20 mg/kg, intravenous, bolus) at 3 min prior to onset of reperfusion, significantly suppressed the reperfusion-induced increase in 8-OH-dG levels. These results suggested that one of the possible protective effect of α-tocopherol acetate was to reduce oxidative DNA damage during in myocardial ischemia and reperfusion.

Effects of lysophosphatidylcholine on the production of interstitial adenosine via protein kinase C-mediated activation of ecto-5'-nucleotidase.

1. Adenosine plays a crucial role in the evolution of ischemic preconditioning. With the use of microdialysis techniques in in situ rat hearts, we assessed the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production), and examined the effects of lysophosphatidylcholine (LPC) on the production of interstitial adenosine. 2. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP, 100 microM). With this system, the dialysate adenosine originates from the dephosphorylation of AMP, catalyzed by endogenous ecto-5'-nucleotidase. The level of dialysate adenosine is a measure of the ecto-5'-nucleotidase activity in vivo. 3. LPC at concentrations of 25 and 50 microM significantly increased the level of dialysate adenosine to 122.7+/-4.3% (n=4, P<0.05) and 158.6+/-7.2% (n=5, P<0.05) of the control, respectively. Chelerythrine (200 microM), a protein kinase C (PKC) inhibitor, completely abolished the increase of dialysate adenosine afforded by LPC (50 microM) (n=5). 4. These data provide the first evidence that LPC does increase the concentration of interstitial adenosine in rat hearts in situ, through the PKC-mediated activation of endogenous ecto-5'-nucleotidase.

Analysis of factors influencing reperfusion-induced arrhythmia in the anesthetized rat heart: the influence of ischemic zone size and hemodynamics

The influence of ischemic zone size and hemodynamics on the reperfusion-induced arrhythmia in the anesthetized rat heart was examined. The heart was subjected to regional ischemia for 4 min by the occlusion of LAD followed by reperfusion for 7 min. After the reperfusion, 64% of the rats died due to irreversible VF. The size of the ischemic zone, the increase in heart rate during ischemia and the incidence of ischemic arrhythmia (VPBs and/or VT) of the animals that died after the reperfusion were significantly greater or higher than those of the surviving animals. There were positive correlations between the size of the ischemic zone and the increase of heart rate during ischemia and the incidence of ischemic arrhythmia. These results indicate that the size of the ischemic zone is a main factor that determines the degree of reperfusion-induced arrhythmia and also contributes to the increase in heart rate and the occurrence of arrhythmia during ischemia. Therefore, in addition to monitoring the blood pressure, heart rate and electrocardiogram, measurement of the size of the ischemic zone is essential for the evaluation of drug effects on reperfusion-induced arrhythmia using this animal model.